It is known that fluorescence illumination in TIRF microscopy is specifically designed such that a total reflection occurs at the cover glass. This only excites objects, which are located directly at the cover glass. Subjacent objects are not reached and can therefore not emit interfering background light. Therefore, TIRF microscopy also provides extremely high-contrast images. However, these images immediately lose their brilliance when a veiling glare occurs. In particular, a veiling glare also excites the object spheres, which are supposed to be suppressed. A veiling glare occurs wherever the excitation light is reflected through lens surfaces and lens and frame edges. The reflections of the lens surfaces can be effectively dealt with through high-quality coating. Reflections, which originate from the lens edges and the frame, can, to a great extent, not be avoided. It is merely possible to ensure that no light reaches said places in the first place. Therefore, the objective must be provided with an extremely high numerical aperture, so the excitation light passes as far away as possible from the lens edges and frames.
Since the excitation light is almost completely reflected at the cover glass, only a very small portion can be used for fluorescence. In order to nonetheless produce bright images, it is sensible to work with high magnifications, focusing the illumination on very small fields. However, this is not the case in confocal applications. Here, the resolution of the objective, irrespective of the aperture on the image side, can be exploited to the fullest. With these applications it is therefore sensible to work with objectives with high aperture and a smaller magnification because a small magnification is equivalent to a large object field and a bothersome objective change can therefore be foregone.
DE 10 2005 051 025 A1 describes a high-aperture, optical imaging system, particularly for microscopes, wherein an immersion objective comprising three optical partial systems with a magnification of less than or equal to 40× and a numerical aperture of greater than or equal to 1.0 and which is chromatically corrected up to the near infrared.
Such a solution is disadvantageous because the frequently desired numerical aperture of 1.4 is not reached.